| Author | Message | ||
     Susanne Wrang Bruun (Swb) |
Hi I'm looking at second derivative spectra of protein powders, which have absorbed different amounts of water. I want to detect interactions and structure changes in the proteins. But have can I interpret a second derivative spectrum ? Do the intensities in second derivatives still reflect the concentration? Do I need MSC before the derivation ? I have identified some amide combination bands, and find that the intensity of some peaks is much more affected by the water than other peaks. What could be the reason for that ? Thanks for your help. Regards Susanne | ||
| David W. Hopkins (Dhopkins) |
Susanne, This sounds like an interesting project. The second derivative is excellent for removing problems with sloping and curving baselines. The nice feature of (any) derivative is that the signal at any wavelength is still proportional to concentration, just as the original absorbance band is. However, if there are multiplicative scatter effects in the scan, they are still present to the same degree in the derivatives. Therefore, I like to use MSC or SNV after the derivative pretreatment to minimize the scattering effects. The first derivative has both a negative and a positive band that can give quantitative information. The second derivative has 2 smaller positive bands as well as the main negative band. This complexity also makes derivatives harder to explain, when there are multiple, overlapping bands for 2 or more materials or groups. I don't have any experience with hydration effects, but there are several opportunities for hydrogen bond formation between the amide group and water. I believe there is a lot of work on determining alpha helicity and beta sheet structure of proteins in the MIR, so I would expect this information to be present in the NIR. You may want to review the MIR to obtain hints on where you could expect this information in the NIR. Best wishes, Dave | ||
| Kathryn Lee |
Susanne, Here is a reference that you might find useful: Barton, F.E., D. S. Himmelsbach, A. M. McClung, E. L. Champagne, Two-Dimensional Vibration Spectroscopy of Rice Quality and Cooking. Cereal Chem., 2002. 79(1): p. 143Ð147. It inclueds NIR, mid-infrared, and Raman spectra of milled and cooked samples of rice. These spectra were regressed two regions at a time by a two-dimensional technique to correlate the spectral regions. The relationships demonstrated that it is possible to recognize the hydration effects caused by gelatinization (cooked samples vs. milled rice). Three water (O-H stretch) spectral bands (960, 1445, 1,930 nm) in the NIR indicated that there were additional phenomena occurring besides the addition of water. Changes in both C-O-H and N-H bands indicated that water was interacting with both starch and protein. The Raman spectra, which were relatively insensitive to water (O-H stretch), revealed only changes in protein that could be associated with denaturization. Though that information may not be exactly relefant to what you are looking for, it should provide some valuble insight into water/protein interactions. Kathryn Lee | ||
| hlmark |
Susanne - One or two points I can contribute: 1) Oftentimes, taking a derivative will hide some information that is present in the original spectrum. I recommend, therefore, that while the derivative is useful for calibration purposes, for visual examination to get insight you should also look at the spectrum itself. 2) Woody Barton and Dave Himmelsbach did their two-dimensional correlation work using a program written in Array Basic for Galactic (now Thermo Informatics group) Industry's GRAMS. If you have GRAMS (or the new GRAMS-AI) I believe the Array Basic program is public domain (since Woody and Dave are with the USDA) and should be avialable. I know I used to have a copy (I just don't know if I can find it now) but several people had copies and someone should be able to give you one if you can't get it from the authors. Howard \o/ /_\ | ||
| Vitas Svedas |
Would like to return to derivatives. To mine mind, second derivatives complicate interpretation of bands (and molecular interactions). For each peak, derivatization produces three lobes from the starting one (as Dave wrote here). Here I inserted picture of 2ndDer of simulated asymmetric Gaussian band (let it succeed to expose).  The positive lobes interfere with negative lobes of neighboring peaks, so separation (and interpretation) problems may appear. Your question about MSC, Susan. Answer depends on how wide is the range of adsorbed water content. Scattering variation goes with strong variation of water content (pouring water into powder). Then you have to apply MSC, SNV or the other scatter correction. If you moisten powder by air humidity increase, the scattering can be assumed constant (it is true for cellulose fiber, at least). Then try the least-squares fit deconvolution of spectra to primary spectral shapes (Gauss�s, Lorentz�s) without derivation and scatter correction. Ideally, these shapes (positions and spectral widths) indicate the effected molecular groups and interactions. I do not know how this deconvolution works in reflection, but in transmission does. Vitas |